Functional And Mechanistic Studies Of IRF9 Regulating SASP Expression During Epithelial Cell Senescence

In most multicellular organisms,aging refers to the gradual and irreversible decline of organ function in the body with age.Cellular senescence is a key biological feature of organismal aging.In senescent cells,cell cycle arrest and its metabolic function is altered,producing senescence associated secretory phenotype(SASP),which is an important factor of the development of many aging-related chronic diseases.Cancer is a great health challenge for human in the 21 st century.And malignant tumors mostly derive from epithelial cells,and cellular senescence is an important obstacle for tumorigenesis.However,studies on the mechanisms of senescence have mainly focused on mesenchymal cells,and the mechanisms of senescence in epithelial cells remain obscure.Multiple arguments imply that epithelial cells have a distinct senescence program from mesenchymal cells.Expression of I-IFN-induced genes increases in aging tissues and organs,and interferons are induced in mesenchymal cells during senescence.The main questions of this study include: what are the characteristics of SASP expression in senescent mammary epithelial cells,whether IFNs are expressed in senescent epithelial cell,and how IRF9 participates in the regulation of SASP induction mechanism.These questions are what we want to focus on and carry out in-depth research,aiming to partially elucidate the senescent mechanism of epithelial cells through this study.And open up a new vision for revealing the early occurrence of breast cancer.In order to investigate the expression characteristics of SASP in senescent epithelial cell,we successfully constructed three senescent models of mammary epithelial cell by stress stimulus.And found that IFNβ and IFNλs spike a surge expression in mammary senescent epithelial cell by RNA-Seq,q PCR and WB,and this high expression of IFNs was a persistent SASP molecule without stress specificity.Mechanistically,by shRNA knockdown screening,we found that the induction of SA-IFNs was dependent on STING and its translocation activation,and although activation of STING is dependent on cGAS during natural immunity,cGAS can recognize exogenous pathogen DNA or endogenous damaged DNA,the expression of SA-IFNs during this process appeared to be independent of cGAS.Subsequently,in order to further investigate the effect of SA-IFNs on senescent epithelial cell.We knocked down and knocked out the key signal transduction molecule,IRF9,which is downstream of IFNs in mammary epithelial cells.We unexpectedly discovered that IRF9 is retro-located upstream to regulate the surge expression of SA-IFNs.Mechanistically,we found that cellular native IRF9 collaborated with STAT2 to regulate the expression of SA-IFNs in an ISRE non-dependent manner.Deletion of IRF9 specifically inhibited the expression of IFNs mediated by STING,but had no significant effect on the expression of early IFNs mediated by pathogenic RNA.In summary,our study identified a unique secretory phenotype in senescent epithelial cells and partially revealed the mechanism,discovered and elucidated the "non-canonical" function of IRF9.IRF9 can regulate the expression of IFNs in senescent HME.These findings provide a potential target for anti-senescence therapy based on cellular senescence intervention strategy,and provide new clues for elucidating the mechanism of early cancer development.